As reviewed by Joseleau and Gancet (Svensk Papperstidning; R123 (1981)), the cohesion of the plant cell wall is primarily due to the presence of its principal components; a crystalline polymer, cellulose, and a three-dimensional macromolecule, lignin, comprising a lignocellulosic material. These components are embedded in a matrix of pectic and hemicellulotic polysaccharides of various nature. It is generally accepted that the relations that exist between these different polymers are established through linkages of different chemical nature. For instance, blocks of lignin are associated through hemicellulose chains. The hemicellulose, another major component of lignocellulosic material, consists largely of 4-O-methylglucuronoxylan, which includes the .beta.-1,4-linked polymer of D-xylose, and herein referred to as xylan. Generally, hardwood pulps contain larger amounts of xylan than do softwood pulps. Such xylan can be enzymatically hydrolyzed to xylose by an endo-xylanase, .beta.-1,4-D-xylan xylanohydrolase, denoted EC 3.2.1.8, and a xylosidase, .beta.-1,4-D-xylohydrolase, denoted EC 3.2.1.37, as discussed by Bernier et al. (Biotechnol. Letters; 7 (11), 797(1985)).
Surprisingly, we have now found that the partial or total digestion of xylan contained in lignocellulosic materials through the use of endo-xylanases, as hereinafter described, provides an attractive alternative to the totally mechanical and/or chemical process for the production of pulps having improved pulp properties such as brightness index. We have found that the production of enzymes suitable for providing improved pulp properties through enzymatic treatment, which production is often quite low in naturally occurring microorganisms, has to be enhanced, in order to be commercially feasible. Further, because release of lignin by hemicellulose cleavage has to be specific in order to prevent the deterioration of certain pulp qualities, e.g. viscosity, that arise due to cellulose hydrolysis, a substantially cellulase-free xylanase mixture has to be used.
Thus, by the molecular cloning and modification of the expression levels of genes coding for endo-xylanase, improved production of such enzymes that are substantially cellulase-free has been attained, and found to be of value for pulp treatment.
The techniques of recombinant genetic technology are known and usually comprise, at their simplest, the steps of obtaining from a donor cell the DNA fragment or gene coding for a desired metabolite or enzyme, cleaving a suitable vector plasmid to provide an insertion site wherein the aforesaid DNA fragment may be inserted into the vector plasmid, inserting the aforesaid DNA into the vector to form a hybrid plasmid, genetically modifying a host bacterium by introducing the hybrid plasmid carrying the aforesaid DNA fragment into the host and culturing the resulting genetically modified (recombinant) bacterium to cause it to produce the desired metabolite.
Molecular cloning of the gene coding for xylanase production has been accomplished in a heterologous system (Bernier, R. et al., Gene; 26, 59 (1983)), wherein the donor strain for the chromosomal DNA was a microorganism of the species Bacillus subtilis and the required DNA fragment (xylnase gene) was cloned and expressed in Escherichia coli as host. The resultant recombinant Escherichia coli was cellulase-negative but the disadvantages associated with such systems include; low levels of protein or enzyme production, gene instability and limited extracellular secretion of the enzyme.
Iwasaki et al. (J. Antibiotics; 39(7), 985 (1986)) reports the molecular cloning of a xylanase gene from the donor strain Streptomyces sp. No. 36a in a homologous system using Streptomyces lividans TK21 as host. The vector plasmid into which aforesaid gene is inserted is pIJ702. The transformants harboring the newly constructed plasmids produce about 10 to 70 times higher levels of extracellular xylanase than that of the donor strain. However, there is no indication of having removed the cellulase activity in the transformants and it is known that under natural circumstances microorganisms of the species Streptomyces lividans produce cellulase.
As a means for the production of an enzymic protein which: 1) exhibits a hydrolase activity, mainly an endo-.beta.-D-xylan hydrolase activity; 2) is cellulase-free 3) is overproduced and present in the supernatant during the growth of a recombinant Streptomyces lividans microorganism (extracellularly secreted) and 4) is soluble in water; the present invention hereinafter defined provides a gene system coding for such an enzyme.
In the treatment of pulps with cellulase-free xylanase that is present in the supernatant in which a suitable microorganism is grown, the cleavage of the hemicellulose will liberate residual lignin present after pulping, and facilitate its partial removal. The removal of lignin is normally associated with a decrease in the Kappa number of the pulp and may be accompanied by an increase in the brightness. A third pulp parameter, viscosity, is normally considered to be a reflection of the extent of cellulose degradation. Viscosity is affected by the degree of crystallinity and the nature of the intermolecular bonding.
French Patent Application No. 2,557,894 (published 1985) discloses a process for treating a hardwood bleached sulphite chemical paper pulp with an enzymatic solution containing xylanase. Particularly large amounts of enzyme are required for the treatment of the bleached pulp in order to impart the effect of relaxation of the pulp fiber structure, which results in the benefit of reducing the amount of subsequent refining required for papermaking. Further, the xylanase secreted by the basidiomycete mushroom Sporotrichum dimorphosporum for use in the reduction of refining, was not cellulase-free and the detrimental cellulase activity was found to be suppressed by the addition of mercuric chloride in the process. However, due to the known toxic and other harmful effects associated with exposure to mercury-containing compounds, their use in removing or inactivating the cellulase enzymes constitutes a distinct disadvantage.
The application of xylanase on hardwood and softwood Kraft pulp for the purpose of brightness improvement and Kappa number reduction upon subsequent bleaching is discussed by Viikari et al. (Proceedings of the International Symposium On Wood and Pulping Chemistry, Paris (1987)). The xylanases are obtained by fermentation of a strain of fungus of the species Aspergillus awamori or by fermentation of bacterial strains of Streptomyces olivochromogenes or Bacillus subtilis. The xylanases exhibit both endo-xylanase and xylosidase activities except the xylanase from the latter bacteria that is xylosidase-free. The enzyme preparations contain traces of cellulase activity. A small brightness increase is observed with either the hardwood or softwood pulp after peroxide delignification following the enzymatic treatment of from 1.0 to 3.4 brightness points, as compared to the brightness achieved in appropriate control experiments, depending on the source of xylanase. The Kappa number reduction for peroxide delignification following enzymatic treatment, as compared to appropriate control experiments, is from 3% to 16% for softwood pulp and from 9% to 22% for hardwood pulp. In many instances, the resulting pulp viscosities were preserved or only slightly lower. There is no indication as to the effect of the enzyme treatment alone on these pulp properties. It is evident, however, that xylanase preparations from different microorganisms impart different effects on pulp properties.
Chauvet et al. (Proceedings of The International Symposium On Wood And Pulping Chemistry, Paris, p. 325, (1987)) report on the use of a xylanase preparation obtained from the basidomycete mushroom Sporotrichum dimorphosporum for use as a pretreatment for conventional chemical pulp bleaching. The crude enzymatic complex is treated in a way that inactivates all polysaccharidase except xylanase activities. The pulp pretreatment comprises an enzymatic treatment followed by washing and subsequent aqueous acid soaking that results in up to a 14% Kappa number reduction for the hardwood sample. The pulp strength is not modified by xylanase in this example.